Haemoproteus infections (Haemosporida,Haemoproteidae) kill bird-biting mosquitoes

Haemoproteus parasites (Haemosporida, Haemoproteidae) are widespread; some species cause severe diseases in avian hosts. Heavy Haemoproteus infections are often lethal for biting midges (Ceratopogonidae), which transmit avian haemoproteids, but there is no information regarding detrimental effect on other blood-sucking insects. We examined effects of Haemoproteus tartakovskyi (lineage hSISKIN1), Haemoproteus lanii (lineages hRB1and hRBS2) and Haemoproteus balmorali (lineage hCOLL3) on the survival of Ochlerotatus cantans, a widespread Eurasian mosquito. Wild-caught females were infected by allowing them to feed on naturally infected birds with light (0.01 %) and high (3.0–9.6 %) parasitaemia. Mosquitoes fed on uninfected birds were used as controls. Both experimental and control groups were maintained under the same laboratory conditions until 20 days post-exposure (dpe). Dead insects were counted daily and used for parasitological examination and PCR-based testing. No difference was discernible in the survival rate of control mosquitoes and those fed on meal with light parasitaemia. There was a highly significant difference in the survival rate between the control group and all groups fed on meals with high parasitaemia, with the greatest mortality reported 1–3 dpe. For 4 dpe, the percentage of survived control mosquitoes (88 %) was 2.2-, 3.6- and 4-fold greater than that of groups fed on meals with high parasitaemia of H. balmorali, H. tartakovskyi and H. lanii, respectively. Numerous ookinetes were observed in the gut area and adjacent tissues located in the head, thorax and abdomen of infected insects 0.5–1 dpe. The migrating parasites damage organs throughout the entire body of mosquitoes; that is the main reason of mortality. To the end of this study, 46 % of mosquitoes survived in control group, but the survival rates of experimental mosquitoes fed on meals with high parasitaemia were between 2.6- and 5.8-fold lower. This study indicates that widespread Haemoproteus infections are markedly virulent for bird-biting mosquitoes, which rapidly die after feeding on heavily infected blood meals.     

Abortive long-lasting sporogony of two Haemoproteus species (Haemosporida, Haemoproteidae) in the mosquito Ochlerotatus cantans, with perspectives on haemosporidian vector research

Haemoproteus spp. are cosmopolitan vector-born haemosporidian parasites, some species of which cause diseases in non-adapted birds. Recent polymerase chain reaction (PCR)-based studies have detected mitochondrial cytochrome b gene lineages of these Haemoproteus parasites in blood-sucking mosquitoes and speculated about possible involvement of these insects in transmission of avian haemoproteids. However, development of Haemoproteus lineages has not been documented in mosquitoes. We infected 304 individuals of Ochlerotatus cantans, a widespread Eurasian mosquito, with Haemoproteus tartakovskyi (lineage hSISKIN1) and Haemoproteus balmorali (lineage hROBIN1). Mosquitoes were allowed to take non-infected and infected blood meals and maintained in the laboratory until 17 days post-infection (dpi). They were tested for presence of sporogonic stages by microscopic and PCR-based methods. Microscopic examination revealed partial development of both parasites in the infected insects. Numerous ookinetes were seen in the gut area and adjacent tissues located in the head, thorax and abdomen of mosquitoes between 1 and 5 dpi. Numerous oocysts were seen in the midgut wall between 4 and 15 dpi; they were also present in the head and thorax of infected mosquitoes testifying to the active movement of ookinetes throughout the body. Oocysts degenerated between 11 and 17 dpi. Sporozoites were not seen in oocysts or mosquito salivary glands, indicating abortive sporogonic development at the oocyst stage. In accordance with microscopy data, PCR and sequencing revealed presence of the lineages hSISKIN1 and hROBIN1 in experimental mosquitoes as long as 15 and 17 dpi, respectively, demonstrating relatively long survival of Haemoproteus parasites in the resistant insects without DNA degeneration. The present study shows that PCR-based diagnostics should be carefully used in vector studies of haemosporidians because it detects parasites in insects for several weeks after initial infection, but does not distinguish abortive parasite development. Demonstration of infective sporozoites in insects is essential for definitively demonstrating the insects are vectors.     

First data on the genetic diversity of avian haemosporidians in China: cytochrome b lineages of the genera Plasmodium and Haemoproteus (Haemosporida) from Gansu Province

A total of 76 birds belonging to 23 species and 14 families was examined for the presence of Plasmodium spp. and Haemoproteus spp. Birds were trapped at four localities in Gansu Province, China, in June–July 2011. DNA was isolated from blood samples and parasite detection, and identification was based on PCR assays and sequences of 479 bp of cyt b gene. The total prevalence of haemosporidians was 21.0 %. Haemoproteus spp. were detected in 14 birds (prevalence 18.4 %). The lineage CYAPIC1 from Cyanopica cyanus, Parus major, Passer montanus and Pyrrhocorax pyrrhocorax was new; it is genetically distinct and probably represents a new species of the genus Haemoproteus. Three lineages represented known species: RBS4 (from Lanius tephronotus), a lineage of Haemoproteus lanii; COLL2 (from Turdus mupinensis), a lineage of Haemoproteus pallidus and TURDUS2 (from Turdus rubrocanus), a lineage of Haemoproteus minutus. The lineage RBS5 (from Lanius cristatus and L. tephronotus) differs by 1.4 % from RBS4 and probably represents an intraspecific entity of H. lanii. The lineages TUCHR1 (recorded from T. mupinensis), WW1 (recorded from Upupa epops) and YWT2 (recorded from Motacilla flava) have not been linked to any known species for the moment. Only one bird was positive for Plasmodium (prevalence 1.4 %), i.e. P. major infected with the lineage GRW4 of Plasmodium relictum. The latter lineage has been considered by previous studies as typical for migratory birds and having transmission in tropical areas only; its record in a sedentary bird in China suggests its transmission in temperate latitudes.     

Avian haemosporidians in haematophagous insects in the Czech Republic

The degree to which avian haemosporidian parasites can exploit different vectors as a definitive host has ecological implications for their transmission and biogeography. Studies targeting haemosporidian parasites using precise molecular detection methods are almost lacking in Central Europe, however. Here, we utilized PCR-based molecular methods to detect avian haemosporidians in insect vectors in the Czech Republic. Nine lineages of parasites belonging to three genera, Haemoproteus, Plasmodium, and Leucocytozoon, were detected in pooled samples of insect individuals, of which three lineages had not yet been discovered in previous studies. All three Leucocytozoon lineages were found exclusively in black flies, while five Haemoproteus lineages were found in biting midges. The most abundant insect species Culicoides kibunensis harbored three Haemoproteus lineages, and the second-most numerous species Culicoides segnis even four. The positive mosquitoes of Culex pipiens complex hosted two parasite lineages, one Plasmodium and one Haemoproteus, the latter of which, however, could suggest the aberrant development of this parasite in an unusual invertebrate host. The co-occurrence of Haemoproteus ROFI1 and TURDUS2 lineages in both insects and birds at the same study plot suggests a transmission of these lineages during breeding season of birds.     

Molecular characterization and phylogenetic analysis of H3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China

In 2013, 15 avian influenza viruses (AIVs), H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2), were isolated from domestic ducks in Zhejiang Province in China. These strains were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. Analysis of the neuraminidase (NA) gene indicates that a re-assortment event between H3 and H9N2 AIV occurred in these ducks. The molecular markers analyzed over the genome of all viruses indicated that these strains were low-pathogenic AIVs. Although there was no evidence of re-assortment in subtype H3 AIVs among the avian species’ and mammalian hosts in this study, continued surveillance is needed considering the important role of domestic ducks in AIV re-assortment.     

Hemoprotozoa in free-ranging birds from rural areas of Mazandaran Province, northern Iran

A range of protozoa infect the blood of birds including Plasmodium, Aegyptianella, Haemoproteus, and Leukocytozoon and are transmitted by known vectors. The parasites occur worldwide and can cause reduced production, anemia, and even death. The aim of present study was to undertake the first survey of hemoprotozoa in the native birds of the Central zone of Mazandaran province. A total of 213 blood samples were collected from five different species of birds, 70 (32.9%) were infected by at least one parasite. The prevalence of infection by Aegyptianella pullorum, Plasmodium spp., and Haemoproteus spp. was 23.9%, 2.3%, and 6.6%, respectively. Our results showed a high prevalence of A. pullorum (38.3%) among ducks, as well as a high prevalence of Haemoproteus spp. (87.5%) in pigeons. Moreover, only ducks and turkeys were found to be infected with Plasmodium spp. Infections with multiple genera were found in 30.04% (64/213) of birds examined which included 91.4% of the 70 infected birds. It also seems that birds which migrate to the area annually might play an important role in infecting domestic birds in the province.     

Wood ducks (Aix sponsa) as potential reservoirs for avian influenza and avian paramyxoviruses

Influenza A viruses (IAVs) and avian paramyxoviruses (APMVs) are important pathogens of poultry worldwide, and both commonly occur in wild waterfowl, especially ducks in the family Anatidae. Although wood ducks (Aix sponsa) are members of the Anatidae, their behaviour differs from most other species in this family, which could affect the transmission of IAVs and APMVs. We collected cloacal and oropharyngeal swab and blood samples from more than 700 wood ducks across nine states in the eastern United States of America. No IAVs were isolated, and based on blocking enzyme-linked immunoassay ELISA results, antibodies to IAVs were only detected in 0.2% of samples. In contrast, 23 (3%) APMVs were isolated (22 Newcastle disease virus and 1 APMV-6), and antibodies to multiple serotypes of APMVs were detected in more than 60% of the samples. After-hatch-year birds were more likely to be antibody positive for APMV-4 and APMV-6 compared to hatch-year birds. Female birds were more likely to be antibody positive for APMV-4 than were male birds. Our results indicate that wood ducks are probably not an important host for IAV but are frequently infected with APMVs.     

Description of Leucocytozoon quynzae sp. nov. (Haemosporida,Leucocytozoidae) from hummingbirds,with remarks on distribution and possible vectors of leucocytozoids in South America

We describe Leucocytozoon quynzae sp. nov. (Haemosporida, Leucocytozoidae), which is the first Leucocytozoon parasite identified to species level in hummingbirds. It was found in the Amethyst-throated Sunangel (Heliangelus amethysticollis, Trochilidae, Apodiformes) captured in the Palacio Forest, which belongs to the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2,900 m above sea level where the transmission occurs; the new species were found both in the high Andean forest and Paramo ecosystem. This parasite is described based on the morphology of its blood stages, a fragment of the mitochondrial cytochrome b gene, and the complete mitochondrial genome. Illustrations of blood stages of the new species are given, and the phylogenetic analysis places this lineage in a well-supported clade with other lineages of unidentified to species level leucocytozoids reported in the Trochilidae birds elsewhere. The new species possess gametocytes in roundish host cells; it can be readily distinguished from other similar leucocytozoids, primarily due to (1) a comma-like shape of the host cell nucleus, which extended one half or less of the circumference of the gametocyte and (2) a large number of prominent volutin granules in the cytoplasm. Identical mitochondrial cytochrome b sequence of Leucocytozoon quynzae was found in different hummingbird species at the type locality and also was reported in one passerine bird at the highlands of Peru. Leucocytozoon quynzae is the first leucocytozoid parasite described from South American birds; its transmission occurs both at low temperatures and high elevations. We discuss some patterns of distribution of avian leucocytozoids in South America and the role of Gigantodax spp. (Diptera, Simuliidae) as potential vectors of Leucocytozoon parasites in the Andean Region.     

Sarcocystis calchasi sp. nov. of the domestic pigeon (Columba livia f. domestica) and the Northern goshawk (Accipiter gentilis): light and electron microscopical characteristics

A novel highly pathogenic Sarcocystis species has been shown to cycle between the Northern goshawk (Accipiter gentilis) as definitive host and the domestic pigeon (Columba livia f. domestica) as intermediate host. However, genetically based characteristics are only available from very few bird-infecting Sarcocystis species. We therefore further characterised morphological properties of this protozoan in both hosts. Using light and electron microscopy, oocysts and sporocysts as well as schizonts and sarcocysts were characterised and compared with available morphological features of previously reported Sarcocystis species of Northern goshawks, Columbidae and genetically closely related species of other avian hosts. Sporocysts shed from day?6 on after experimental infection by the Northern goshawk were of ovoid appearance (11.9?×?7.9 μm). Ultrastructurally, schizonts of all developmental stages were found in the liver, spleen and next to or in endothelial cells of various organs of domestic pigeons 7 to 12 days after experimental infection. The cyst wall surface of slender sarcocysts (1 to 2 mm in length and 20 to 50 μm in width) was smooth and lacked protrusions. Cystozoites were lancet-shaped and measured 7.5?×?1.5 μm in Giemsa stain smears. The morphological findings, when combined with data of experimental infection and genetic studies, convergently indicate that the recently discovered Sarcocystis species represents a new species. We therefore propose to name this parasite Sarcocystis calchasi species nova.     

Description and molecular characterization of Plasmodium (Novyella) unalis sp. nov. from the Great Thrush (Turdus fuscater) in highland of Colombia

Plasmodium (Novyella) unalis sp. nov. was found in the Great Thrush, Turdus fuscater (Passeriformes, Turdidae) in Bogotá, Colombia, at 2,560 m above sea level where the active transmission occurs. This parasite is described based on the morphology of its blood stages and a fragment of the mitochondrial cytochrome b gene (lineage UN227). Illustrations of blood stages of new species are given, and the phylogenetic analysis identifies closely related species and lineages of avian malaria parasites. The new species is most similar to Plasmodium (Novyella) vaughani (lineage SYAT05), a cosmopolitan avian malaria parasite; these parasites are also closely related genetically, with a genetic difference of 3.2 % between them. P. unalis can be readily distinguished from the latter species morphologically, primarily due to the (1) presence of a single large, circular shaped pigment granule in the erythrocytic trophozoites and meronts; (2) presence of prominent vacuoles in trophozoites and growing meronts; and (3) presence of predominantly fan-like shaped erythrocytic meronts. Cytochrome b lineages with high similarity to the new species have been reported in Costa Rica, Brazil, Chile, and USA. It is probable that the new species of malaria parasite is widely distributed in the New World. This parasite has been reported only in the Great Thrush at the study site and might have a narrow range of avian hosts. Records of P. unalis are of particular theoretical interest due to its active transmission at highlands in Andes. Possible influence of urbanization on transmission of this malaria parasite in Bogotá is discussed.     

Molecular characterization of five widespread avian haemosporidian parasites (Haemosporida), with perspectives on the PCR-based detection of haemosporidians in wildlife

Haemosporidians (Haemosporida) are cosmopolitan in birds. Over 250 species of these blood parasites have been described and named; however, molecular markers remain unidentified for the great majority of them. This is unfortunate because linkage between DNA sequences and identifications based on morphological species can provide important information about patterns of transmission, virulence, and evolutionary biology of these organisms. There is an urgent need to remedy this because few experts possess the knowledge to identify haemosporidian species and few laboratories are involved in training these taxonomic skills. Here, we describe new mitochondrial cytochrome b markers for the polymerase chain reaction (PCR)-based detection of four widespread species of avian Haemoproteus (Haemoproteus hirundinis, Haemoproteus parabelopolskyi, Haemoproteus pastoris, Haemoproteus syrnii) and 1 species of Plasmodium (Plasmodium circumflexum). Illustrations of blood stages of the reported species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. This study indicates that morphological characters, which have been traditionally used in taxonomy of avian haemosporidian parasites, have a phylogenetic value. Perspectives on haemosporidian diagnostics using microscopic and PCR-based methods are discussed, particularly the difficulties in detection of light parasitemia, coinfections, and abortive parasite development. We emphasize that sensitive PCR amplifies more infections than can be transmitted; it should be used carefully in epidemiology studies, particularly in wildlife parasitology research. Because molecular studies are describing remarkably more parasite diversity than previously expected, the need for traditional taxonomy and traditional biological knowledge is becoming all the more crucial. The linkage of molecular and morphological approaches is worth more of the attention of researchers because this approach provides new knowledge for better understanding insufficiently investigated lethal diseases caused by haemosporidian infections, particularly on the exoerythrocytic (tissue) and vector stages. That requires close collaboration between researchers from different fields with a common interest.     

First survey of the occurrence of duck enteritis virus (DEV) in free-ranging Polish water birds

Duck plague (DP) caused by anatid herpesvirus 1, also called duck enteritis virus (DEV), presents one of the most important concerns in mass waterfowl production. Apart from geese and ducks, free-ranging water birds are the most notorious infection carriers. The epidemiology of DEV in Western Europe remains unknown. Therefore, it was reasonable to conduct a study on its occurrence using modern but simple real-time loop-mediated isothermal amplification (LAMP). Analysis of 132 field isolates showed the presence of DEV in 96 birds (72.7 %), and it was found predominantly in wild ducks (Anas platyrhynchos) and mute swans (Cygnus olor). This virus was also found in graylag geese (Anser anser), tundra bean geese (Anser fabalis), and grey herons (Ardea cinerea). The results were recorded as green colour of positive samples, fluorescence under ultraviolet light, and florescent curves in a real-time PCR system. This study indicates the high prevalence of DEV among free-ranging water birds in Poland and the possible transmission to other birds settling in the water environment. This is the first report of DEV detection among free-ranging water birds in Poland.     

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, <Emphasis Type="Italic">Falco naumanni</Emphasis>

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.     

Description,molecular characterization,and patterns of distribution of a widespread New World avian malaria parasite (Haemosporida: Plasmodiidae), Plasmodium (Novyella) homopolare sp. nov.

Plasmodium (Novyella) homopolare, a newly described Plasmodium species, was found in a wide range of Passeriformes species in California, USA, and Colombia. This parasite infected more than 20 % of the sampled bird community (N = 399) in California and was found in 3.6 % of birds sampled (N = 493) in Colombia. Thus far, it has been confirmed in North and South America where it is present in numerous species of migratory and resident birds from six families. Based on 100 % matches, or near-100 % matches (i.e., ≤2-nucleotide difference), to DNA sequences previously deposited in GenBank, this parasite is likely also distributed in the Eastern USA, Central America, and the Caribbean. Here, we describe the blood stages of P. homopolare and its mtDNA cytochrome b sequence. P. homopolare belongs to the subgenus Novyella and can be readily distinguished from the majority of other Novyella species, primarily, by the strictly polar or subpolar position of meronts and advanced trophozoites in infected erythrocytes. We explore possible reasons why this widespread parasite has not been described in earlier studies. Natural malarial parasitemias are usually light and co-infections predominate, making the parasites difficult to detect and identify to species when relying exclusively on microscopic examination of blood films. The combined application of sequence data and digital microscopy techniques, such as those used in this study, provides identifying markers that will facilitate the diagnosis of this parasite in natural avian populations. We also address the evolutionary relationship of this parasite to other species of Plasmodium using phylogenetic reconstruction.     

Investigating a crow die-off in January–February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh

We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.     

Haemosporidian parasites of a European passerine wintering in South Asia: diversity, mixed infections and effect on host condition

We studied haemosporidian parasites in the scarlet rosefinch Carpodacus erythrinus in a small isolated semicolony during an eight-year period using molecular methods of parasite detection. The scarlet rosefinch is an interesting model of parasite host species. It winters in South Asia which represents a rare exception among European passerines. Males express yellow to red carotenoid-based plumage ornament which is a good predictor of male reproductive success. In 240 blood samples originating from 199 adult individuals, the total parasite prevalence reached 60 %. Prevalence varied among years from 36 to 81 % in Haemoproteus, 8 to 22 % in Plasmodium, and 0 to 14 % in Leucocytozoon. Twenty parasite lineages were detected (Haemoproteus: 5 lineages, Plasmodium: 10 lineages, and Leucocytozoon: 5 lineages). Among them, the Haemoproteus ROFI2 lineage, which is a host-specific parasite lineage of the scarlet rosefinch, was the most frequently found. Parasite lineages showed varying degree of lineage specificity. While Haemoproteus lineages detected in the scarlet rosefinch have relatively narrow host breadth restricted mainly to Fringillidae family, Leucocytozoon and Plasmodium lineages generally showed wider host range. The presence of some parasite lineages hitherto detected in sedentary European passerines (SISKIN1, CCF3, BT2) or in Culicoides biting midges at the same locality (ROFI1) suggest local transmission. On the contrary, lineages LK05 and FANTAIL1 that were previously reported exclusively from Asian hosts imply parasite transmission at the scarlet rosefinch wintering sites in South Asia. Mixed infections were found in 17 % of infected samples and comprised mainly the most frequent lineages. The pattern of concomitant infections seemed to be rather random and matched expected levels based on lineage frequencies. Between-year comparisons revealed that in a majority of the repeatedly captured individual hosts the infection status remained unchanged (individuals stayed uninfected or possessed the same parasite lineages). However, 16 gains and 8 losses of lineages were also reported. We have not found any effect of haemosporidians on male carotenoid ornament expression or host body mass.     

Genetic diversity of avian blood parasites in SE Europe: Cytochrome <Emphasis Type="Italic">b</Emphasis> lineages of the genera <Emphasis Type="Italic">Plasmodium</Emphasis> and <Emphasis Type="Italic">Haemoproteus</Emphasis> (Haemosporida) from Bulgaria

We used a nested PCR protocol to examine the genetic diversity of cytochrome b (cyt b) lineages from blood parasites of the genera Plasmodium and Haemoproteus in birds in Bulgaria. In total, 460 birds of 43 species and 14 families (mostly passerines) were examined for the presence of infections. Of them, 267 were recognised as infected with haemosporidian parasites. Mixed infections were recorded in 24 individuals (9%). Besides the 24 individuals with mix infections, 114 (43%) were positive for Plasmodium spp. and 129 (48%) for Haemoproteus spp. We identified 52 genetic lineages of haemosporidian parasites: 38 of Haemoproteus and 14 of Plasmodium. Twelve new cyt b lineages of Haemoproteus were recorded; they occurred in the following hosts: grey-faced woodpecker (Picus canus), golden oriole (Oriolus oriolus), jay (Garrulus glandarius), barred warbler (Sylvia nisoria), song thrush (Turdus philomelos), spotted flycatcher (Muscicapa striata), spanish sparrow (Passer hispaniolensis), hawfinch (Coccothraustes coccothraustes), and cirl bunting (Emberiza cirlus). We also detected 22 new host records for previously known lineages. The most common lineage was SGS1 (Plasmodium relictum), which had a total prevalence of 14% and occurred in 8 host species belonging to 5 families. Three of the cyt b lineages of genus Haemoproteus (DURB1, DURB2 and SYNIS2) showed more than 5% divergence from all described morphologically lineages. These lineages probably represent at least 2 different morphospecies which remains to be identified.     

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology,molecular characteristics and phylogeny

The arctic fox (Vulpes lagopus) is a critically endangered species in Norway, and therefore, the small population is closely monitored, and most foxes found dead are subjected to necropsy. In two deceased foxes, thin-walled muscular sarcocysts were first detected in histological sections, and numerous sarcocysts were later found in frozen and thawed muscle samples from Fox 1. These sarcocysts measured 1–12?×?0.1–0.25 mm and had closely spaced, short, knob-like protrusions, giving the cysts a serrated outline. Genomic DNA was extracted from eight isolated sarcocysts (Fox 1) and two muscle samples (Fox 2) and subjected to polymerase chain reaction amplification at four loci: the nuclear 18S and 28S ribosomal RNA genes and internal transcribed spacer 1 region and the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). Both foxes were infected by the same Sarcocystis sp., which displayed little or no genetic variation at the three nuclear loci (99.9–100 % identity) and slightly more variation at cox1 (99.4–100 % identity). Sequence comparisons and phylogenetic analyses revealed that this species was distinct from other named Sarcocystis spp. but was closely related to various species using avian intermediate hosts and possibly identical to an unnamed species reported from two American dogs. The species described from the two arctic foxes was named Sarcocystis arctica n. sp.     

Parrot bornavirus-2 and -4 RNA detected in wild bird samples in Japan are phylogenetically adjacent to those found in pet birds in Japan

Bornaviruses (family Bornaviridae) are non-segmented negative-strand RNA viruses. Avian bornaviruses (ABVs), which are causative agents of proventricular dilatation disease, are a genetically diverse group with at least 15 genotypes, including parrot bornaviruses (PaBVs) and aquatic bird bornavirus 1(ABBV-1). Borna disease virus 1(BoDV-1), which infects mammals and causes neurological diseases, has also been reported to infect avian species, although the numbers of the cases have been markedly fewer than those of ABVs. In this study, we conducted genetic surveillance to detect ABVs (PaBV-1 to -5 and ABBV-1) and BoDV-1 in wild birds in Japan. A total of 2078 fecal or cloacal swab samples were collected from wild birds in 2006, 2007, 2008, and 2011, in two regions of Japan. The results demonstrated the presence of PaBV-2 and -4 RNA, while no positive results for other PaBVs, ABBV-1, and BoDV-1 were obtained. PaBV-2 and -4 RNA were detected in 18 samples (0.9 %) of the genera Anas, Grus, Larus, Calidris, Haliaeetus, and Emberiza, in which either PaBV-2 RNA or PaBV-4 RNA, or both PaBV-2 and -4 RNA were detected in 15 (0.7 %), 5 (0.2 %), and 2 (0.1 %) samples, respectively. The nucleotide sequences of PaBV-2 and -4 detected in these samples from wild birds are phylogenetically close to those found in samples from pet birds in Japan, with identities ranging from 99.8 to 100 % and from 98.2 to 99.4 %, respectively. To the best of our knowledge, this is the first report on the detection of PaBV-2 and -4 RNA detected in samples from wild birds.     

Variation in viral shedding patterns between different wild bird species infected experimentally with low-pathogenicity avian influenza viruses that originated from wild birds

The prevalence of infection with avian influenza (AI) virus varies significantly between taxonomic Orders and even between species within the same Order. The current understanding of AI infection and virus shedding parameters in wild birds is limited and largely based on trials conducted in mallards (Anas platyrhynchos). The objective of the present study was to provide experimental data to examine species-related differences in susceptibility and viral shedding associated with wild bird-origin low-pathogenicity avian influenza (LPAI) viruses in multiple duck species and gulls. Thus mallards, redheads (Aythya americana), wood ducks (Aix sponsa), and laughing gulls (Leucophaeus atricilla) were inoculated experimentally with three wild mallard-origin LPAI viruses representing multiple subtypes. Variation in susceptibility and patterns of viral shedding associated with LPAI virus infection was evident between the duck and gull species. Consistent with the literature, mallards excreted virus predominantly via the gastrointestinal tract. In wood ducks, redheads, and laughing gulls, AI virus was detected more often in oropharyngeal swabs than cloacal swabs. The results of this study suggest that LPAI shedding varies between taxonomically related avian species. Such differences may be important for understanding the potential role of individual species in the transmission and maintenance of LPAI viruses and may have implications for improving sampling strategies for LPAI detection. Additional comparative studies, which include LPAI viruses originating from non-mallard species, are necessary to further characterize these infections in wild avian species other than mallards and provide a mechanism to explain these differences in viral excretion.